There is a broad class of scanning microscopes and mapping systems in which a light source is focused to a point on a specimen, and the light reflected (or emitted) from that point is measured by a detector. An image of the specimen is recorded by scanning the illuminated point across the specimen in a raster scan (scanning beam system), or by moving the specimen in a raster scan under a stationary beam (scanning stage system). Scanning stage microscopes are often used when the specimen is large (for example, when an image is required of a whole semiconductor wafer).
A simple prior art confocal scanning stage laser microscope is shown in FIG. 1. In this implementation the beam from laser 102 is focused by lens 104 onto pinhole 106, and the light passing through pinhole 106 passes through beamsplitter 108 and is focused by objective lens 110 to a focal spot 111 at the surface of (or inside) specimen 112. For best resolution, focal spot 111 should be diffraction limited. Light reflected from or emitted by the specimen at focal spot 111 is collected by objective lens 110, and part of this light is reflected by beamsplitter 108 to be focused at detector pinhole 114. Pinhole 106 and detector pinhole 114 are confocal with focal spot 111. Light passing through detector pinhole 114 is collected by detector 116. Reflected light from focal spot 111 at specimen 112 passes through detector pinhole 114, but light from any other point on the specimen runs into the edges of detector pinhole 114, and is not collected. This gives the confocal microscope increased resolution over a non-confocal microscope, and since detector pinhole 114 rejects light that does not come from the focal plane, gives the confocal microscope its optical slicing ability, which allows it to record true three dimensional images. The microscope shown in FIG. 1 uses scanning stages 118 to move the specimen under the stationary laser beam to record the image, but configurations which scan the beam instead of scanning the specimen are also known. Microscopes using infinity-corrected optics are also common, both in scanning-stage and scanning-beam configurations. These configurations are described in J. Pawley, "The Handbook of Biological Confocal Microscopy" IMR Press, Madison Wis. 53706 (1989) In addition, it is known that detector pinhole 114 and detector 116 behind it (which together comprise a confocal detector) can be replaced with a small detector whose area is the same as that of detector pinhole 114.
Scanning stage microscopes have several disadvantages over scanning beam microscopes. The main disadvantage is the increased time required to acquire an image, because scanning the specimen under a fixed beam is inherently slower than scanning the beam. Rapid scanning of the specimen and stages can also cause vibrations in the microscope and/or in the specimen itself which can cause blurring in the acquired image, an additional disadvantage. One advantage of scanning stage microscopes is their ability to perform very large scans with high spatial resolution. One example of such an application is the use of a non-confocal scanning stage microscope for photoluminescence mapping of semiconductor wafers as described by Moore et al, "A Spatially Resolved Spectrally Resolved Photoluminescence Mapping System", Journal of Crystal Growth 103, 21-27 (1990). When scanning optical microscopes are used with large specimens like semiconductor wafers, they are often referred to as "mapping systems" or "mappers".
A prior-art infinity-corrected scanning beam confocal microscope is shown in perspective in FIG. 2. Light beam 203 from light source 202 is focused on pinhole 208 by lens 206. The expanding beam exiting pinhole 208 is focused to a parallel beam by lens 210. (Lens 206, pinhole 208 and lens 210 constitute a spatial filter and beam expander.) The parallel beam passes through beamsplitter 212 and is deflected in the x-y plane by first scanning mirror 214, which rotates about an axis parallel to the z-direction. Lenses 216 and 218 of focal length f.sub.2 return the deflected light beam to the center of second scanning mirror 220, which rotates about an axis parallel to the x-direction and imparts a deflection in the y-z plane. Lenses 222 and 224 of focal length f.sub.3 return the deflected beam (which now has been deflected by both scanning mirrors) to enter objective lens 226 centered on its entrance pupil. Objective lens 226 focuses the light to a focal spot 227 (which for best resolution should be a diffraction-limited spot) at the surface of or inside specimen 228. The focus position is set by focus stage 230, which moves in the z-direction. Light reflected back from or emitted by the tiny volume of the specimen at focal spot 227 is collected by objective lens 226 and passes back through the scan system of the microscope. Part of this returning beam is reflected by beamsplitter 212 towards lens 232. Lens 232 focuses the light onto detector pinhole 234. Light originating from focal spot 227 in specimen 228 passes through detector pinhole 234 and is detected by detector 236, light from any other point in specimen 228 hits the metal edges around detector pinhole 234, and is not detected. As the scanning mirrors 214 and 220 move focal spot 227 across specimen 228, an image is recorded of features of the specimen that are in the focal plane of objective lens 226. If the specimen position is changed by moving it toward or away from the objective lens, an image from a different slice through the specimen is recorded.
Several other prior art embodiments of the scanning beam confocal reflected light microscope exist, including microscopes using a single mirror that can be scanned about two perpendicular axes, and microscopes using acousto-optic deflectors as described by Pieter Houpt et al in U.S. Pat. No. 4,863,226.
Another prior art embodiment of a confocal scanning-beam laser microscope uses an acousto-optic deflector to scan the beam in the fast scan direction and a scanning mirror to scan it in the slow scan direction. In this embodiment, the reflected or fluorescence light returning from the specimen is descanned by the mirror in the slow scan direction, and is then reflected toward a confocal detector comprising a linear detector array so no descanning is required in the fast direction. This has the advantage of allowing very high speed scans without having to pass the reflected or fluorescence light back through the acousto-optic deflector, which would considerably reduce the intensity of the light reaching the detector.
Yet another prior-art embodiment of a confocal scanning-beam optical microscope is the class of microscopes known as Nipkow Disk microscopes. The microscopes in this class were described by Gordon Kino in "Efficiency in Nipkow Disk Microscopes" in "The Handbook of Biological Confocal Microscopy", p. 93-99 (IMR Press, Madison, Wis. 53706, edited by J. Pawley). These microscopes are different from the microscopes already described mainly in that a large number of incoming scanning beams are focused on the specimen simultaneously, and reflected or fluorescent light beams from these focused spots are detected simultaneously.
Scanning beam microscopes that are not infinity corrected have also been made. All of these microscopes are often used for fluorescence measurements (see J. Pawley, referred to earlier) .
A prior art scanning stage reflection and transmission confocal microscope is shown in FIG. 3. In this microscope transmitted light is collected by a second objective lens 320 placed beneath specimen 112, confocal with the first objective lens 110. Transmitted light from the focal point is collected by second objective lens 320, passes through pinhole 322 in front of transmitted-light detector 324, whereas light from other points in specimen 112 hits the edges of pinhole 322 and does not reach detector 324. The sample is translated in a raster scan relative to the fixed beam. Thus this microscope performs optical image slicing in transmission as well as in reflection. A scanning stage transmission confocal microscope was described by G. J. Brakenhoff, "Imaging Modes in Confocal Scanning Light Microscopy", Journal of Microscopy 117, 233-242 (1979).
The scanning stage transmission microscope shown in FIG. 3 has all of the disadvantages of scanning stage microscopes listed earlier, but this microscope has the advantage of being able to form images in transmission. One disadvantage is that the resolution of both reflection and transmission images becomes progressively poorer as the microscope is focused further beneath the top surface of the specimen. This is caused by spherical abberation in the specimen.
Scanning beam transmission confocal microscopes have been thought to be impractical (see D. Goodman, "Confocal Microscopy", notes from a course at SPIE's 1989 Symposium on Microlithography, San Jose, Calif. (1989)), since they require precise synchronization of two scan systems, one in the source arm of the microscope, and one in the detector arm, so the source pinhole and detector pinhole are imaged at the same point on the specimen as the scan proceeds. However, one design of a transmission scanning beam microscope has been described in the literature by S. Goldstein, "A No-Moving-Parts Video Rate Laser Beam Scanning Type 2 Confocal Reflected/Transmission Microscope", Journal of Microscopy 153, RP1-RP2 (1989) and in U.S. Pat. No. 4,827,125. Goldstein does not descan the transmitted beam; instead his invention uses an Image Dissector Tube to scan the detector pinhole in synchronism with the scanning transmitted (or reflected) beam. Precise synchronization is difficult and requires sophisticated electronics, and different optical paths must be used for reflected-light and transmitted-light imaging, requiring two complete detector systems. The only practical detector that has been used in this microscope is the image dissector tube, severely limiting the choice of detectors for different applications.
The prior art confocal scanning beam microscopes described herein and in the reference literature are used generally reflected-light and fluorescence or photoluminescence imaging. These microscopes have several limitations. First, the image-slicing ability of these confocal microscopes enables them to record three-dimensional images, but viewed from one side of the specimen only. Second, when scanning specimens more than a few microns thick, spherical abberation degrades the image as the beam penetrates deeper into the specimen, and the degradation increases with depth. This is true for both reflected-light and fluorescence or photoluminescence imaging. Third, biological specimens are often only weak reflectors, requiring either high levels of illumination or frame averaging to build up an image. Fourth, when fluorescence or photoluminescence measurements are performed using these microscopes, the focus of the microscope may be at a slightly different position for the incoming illumination than it is for the photoluminescence or fluorescence wavelengths emitted by the specimen, since photoluminescence or fluorescence occurs at wavelengths that are different from the exciting wavelength, and the focal length of a microscope objective varies slightly with wavelength.
It is an object of the present invention to provide a practical scanning beam confocal microscope that will record confocal images in both transmission and reflection.
It is another object of this invention to provide confocal images from both sides of a specimen, in both transmission and reflection, which allows the operator to form a reflected-light image of both the top and bottom of an opaque specimen, thus allowing the microscope to additionally record the bottom half of a three-dimensional image of an opaque specimen. In addition., this can reduce the effect of spherical abberation by allowing the operator to illuminate the specimen with a beam that impinges on the specimen from the side closest to the focal plane. Thus, a beam from the top can be chosen when imaging the top half of a specimen, and one from the bottom when imaging the bottom half.
It is yet a further object of this invention to provide a scanning-beam confocal photoluminescence or fluorescence microscope in which the incoming illumination is focused by one objective lens and the fluorescence or photoluminescence emitted by the specimen is collected by the second objective lens, which now can be placed at the proper focal distance to collect light at the fluorescence or photoluminescence wavelength emitted from the illuminated spot in the specimen.
It is yet a further object of this invention to provide a confocal scanning beam laser microscope or mapping system, that allows the operator to choose any of four operating modes for the microscope: transmission with illumination from the top of the specimen, transmission with illumination from the bottom of the specimen, reflection from the top, and reflection from the bottom, in addition to being able to use the microscope for fluorescence or photoluminescence imaging.
It is a further object of this invention to provide a confocal scanning stage microscope, or one using a scanning stage in one direction and a scanning beam in the other, that will provide any of the capabilities described in the objects stated above.